PROJECT SUMMARY Transplantation tolerance, a state in which immunosuppression can be stopped while grafts remain functional, is a major goal in the field to reduce the morbidity associated with long-term usage of immunosuppression. Tolerance can be spontaneously achieved in rare patients. However, a proportion of tolerant patients lose their grafts after years of tolerance, sometimes following episodes of infections, indicating that tolerance is not always robust and persistent. The goal of Project 1 has been to investigate the mechanisms underscoring tolerance when it is robust and durable. To address this question mechanistically, we used a mouse model of donor-specific transplantation tolerance that we consider robust because it withstands many inflammatory challenges, and tracked graft-reactive T cells using seeded TCR-Tg T cells, or using fluorescent pMHC Class I and Class II multimers to identify endogenous populations. We have found that robust tolerance depends on multiple redundant cell-intrinsic and -extrinsic (constraint of cell numbers, Tregs, engagement of PD-L1) mechanisms to control alloreactive T cells. In this model, infection with Listeria monocytogenes (Lm) 60 days after transplantation eroded the tolerance, in that elimination of single mechanisms of tolerance was sufficient to precipitate graft rejection after, but not before infection. Therefore, our programmatic research revealed that transplantation tolerance is not an all-or-none state but rather reflects a gradation of individual mechanisms of T cell tolerance, each of which may be sufficient to prevent rejection, but several of which together maintain a more robust tolerance. In this Competitive Renewal, we will concentrate on an important property of robust tolerance, namely its resilience, defined as the ability to ?spring back? after a period of stress. Indeed, Lm infection in tolerant hosts precipitated rejection in about 50% of the mice, but tolerance re-emerged when the inflammation subsided, and second donor-matched allografts were spontaneously accepted. This `memory of tolerance' that dominated over a memory of rejection was dependent on regulatory T cells (Tregs) and their suppression of the alloreactive conventional T cells (Tconv) that had not been deleted during the induction of tolerance. Analysis of tolerant Tconv before and after infection revealed 2 novel properties: programmed T cell-intrinsic dysfunction, and the preferential preservation of low avidity graft-reactive T cell clones. We hypothesize that these two features make Tconv more susceptible to regulation, and are thus critical for tolerance to be robust at initiation and to return after transient reactivation by inflammatory events. We propose two aims: Aim 1. To define the molecular characteristics and functional defects of T cell-intrinsic dysfunction; Aim 2. To assess the impact of controlling T cell `avidity maturation' in transplantation tolerance. We anticipate that the completion of these studies will provide insights into the mechanisms underscoring the resilience of tolerance and lead to the identification of new therapeutic approaches to promote or re-induce a state of transplantation tolerance that can persist through inflammatory challenges.